/. Embryol. exp. Morph. 89, 57-70 (1985)
57
Printed in Great Britain © The Company of Biologists Limited 1985
Cell movement in intact and regenerating planarians.
Quantitation using chromosomal, nuclear and
cytoplasmic markers
EMILI SALO AND JAUME BAGUNA
Departament de Genetica, Facultat de Biologia, Universitat de Barcelona, Diagonal
645, 08028 Barcelona, Spain
SUMMARY
One of the tenets of Wolff and Dubois' 'neoblast theory' of planarian regeneration (Wolff &
Dubois, 1948) is that blastema is mainly formed by the accumulation of undifferentiated
parenchymal cells (neoblasts) that can migrate, if needed, over long distances to the wound.
That neoblasts migrate was claimed by these authors after partial X-irradiation, and total Xirradiation and grafting using planarian strains of different pigmentation. From this they
suggested that migration of neoblasts is stimulated by the wound and directed towards it.
To study the nature and extent of such 'migration' in intact and regenerating organisms, and in
order to avoid the flaws of using pigmentation as a marker, we made grafts between sexual and
asexual races of Dugesia(S)mediterranea that differ in a chromosomal marker, and between
diploid and tetraploid biotypes of Dugesia(S)polychroa that differ in nuclear size. Also,
fluorescent latex beads were used as cytoplasmic markers to follow 'migration' of differentiated
cells. The hosts were irradiated or non-irradiated intact and regenerating organisms.
The results show that: 1) movement of graft cells into host tissues occurs in intact organisms at
a rate of = 40|um/day, and that this increases up to =75jum/day in irradiated hosts; 2)
movement of cells occurs evenly in all directions; 3) regeneration does not speed up rate of
movement nor drives cells preferentially to the wound; 4) spreading of cells is mainly due to the
movement of undifferentiated cells (neoblasts); and 5) higher rates of movement are correlated
with higher mitotic indexes. From this, it is concluded that the so-called 'migration' of neoblasts
is not a true cell migration but the result of the slow, even and progressive spreading of these
cells mainly caused by random movements linked to cell proliferation. The implications of these
results for blastema formation and the origin of blastema cells are discussed.
INTRODUCTION
The most widely held theory of blastema formation during planarian
regeneration is Wolff and Dubois' theory of neoblast migration and proliferation
(Wolff & Dubois, 1948). According to it, blastema cells and undifferentiated
parenchymal neoblasts are alike, and these cells, if needed, can migrate over long
distances to the wound. Basically, this follows after experiments of partial X-ray
irradiation and amputation (Dubois, 1949), and experiments of total X-ray
irradiation and grafting (Lender & Gabriel, 1965) where a delay in blastema
formation was observed, delay proportional to the distance from the unirradiated
region (or graft) to the wound. That cells making the blastema came from
Key words: cell movement, cell markers, regeneration, chimaeras, planarian, nuclear,
chromosomal, cytoplasmic, Dugesia spp.
58
E. S A L 6 AND J. BAGUNA
unirradiated tissues and not from revitalized irradiated tissues was shown using
planarian strains of different pigmentation.
From these experiments, two main conclusions arose: 1) blastema cells are
migratory neoblasts coming from unirradiated regions; therefore, neoblasts are
the source of blastema cells and most probably totipotent; and 2) the migration of
neoblasts is stimulated by the wound and directed towards it.
While the first conclusion could be questioned on the grounds that migratory
neoblasts could be the result of cell dedifferentiation within the unirradiated
region (or graft), the second conclusion poses two main questions: 1) if neoblast
migration is stimulated by the wound, we should expect little or no migration of
graft cells within intact unirradiated or irradiated organisms; 2) if neoblasts
migration is directed (oriented) towards the wound, we should expect that
anteriorly regenerating organisms (irradiated or not) bearing a graft will show a
preferential migration of graft cells towards the wound area rather than towards
posterior or lateral regions.
To follow graft cells within a host tissue, strains of different pigmentation have
been widely used. This method, though useful, has several flaws, the main ones
being the long time needed to see a clear result, the difficulties in quantitating
it, and the uncertainties of pigment cells as faithful markers of phenomena
happening in internal tissues. No wonder that in a recent review on cell
movements within organisms, Trinkaus (1984) says rather ironically 'these cells,
called neoblasts, have been assigned full pluripotency and legendary migratory
capacities. They have even been thought to move from one end of a creature to
another, . . . there is certainly no evidence that such cells engage in their
postulated peregrinations', (p. 38, op. cit.).
To overcome these difficulties and criticisms, we have made use of a
chromosomal marker (a heteromorphosis) that characterizes the asexual race of
Dugesia(S)mediterranea as compared to the sexual race of the same species, as well
as the difference in nuclear size between neoblasts of the diploid and tetraploid
biotypes of Dugesia(S)polychroa. Also, fluorescent latex beads, taken up by
specific differentiated cell types, have been used as cytoplasmic markers to track
cell movements.
MATERIALS AND METHODS
Planarians used in this study were as follows: 1) the asexual race of Dugesia(S)mediterranea
(Benazzi, Ballester, Baguna & Puccinelli, 1972) from Barcelona (Spain); 2) the sexual race of
Dugesia(S)mediterranea (Benazzi & Benazzi-Lentati, 1976) from Sardinia (Italy), kindly sent by
Prof. N. G. Lepori; and 3) the biotypes A (2n = 8) and D (4n = 16) of Dugesia(S)polychroa
(Benazzi & Benazzi-Lentati, 1976) from Sardinia (Italy), also sent by Prof. N. G. Lepori. The
organisms were reared in Petri dishes with spring water in the dark at 17 ± 1°C and fed with
Tubifex sp. In all experiments, one-week starved organisms of large size (2*15 mm in length)
were used, and the temperature kept at 17 ± 1°C.
Terminology
Since the classic paper of Wolff & Dubois (1948), authors dealing with movements of cells
within intact and regenerating planarians have employed the term cell migration to refer to the
Cell movement in planarians
1A
59
-"3
Fig. 1. Karyotypes of Dugesia(S)mediterranea. (A) Asexual race (As), having a clear
heteromorphosis (arrow) in the third pair (J) of chromosomes; (B) Sexual race (5),
having chromosomes of equal length.
displacement of cells from a graft to distant sites in the host. However, it is still not clear if, in
planarians, this phenomenon results from an active migration of individual cells (true cell
migration) or from the slow spreading of graft cells within a host due to cell proliferation (cell
repopulation).
Therefore, when referring to the gradual and mutual spreading of cells between graft and
host, we would avoid in this paper the use of the term cell migration speaking instead of cell
spreading or cell movement.
Chromosomal marker
The asexual race (As) of Dugesia(S)mediterranea has an heteromorphosis in the third pair of
chromosomes (Fig. 1A), the long arm of one homologue being much longer than the other
(Baguna, 1973; De Vries, Baguna & Ball, 1984). In contrast, the sexual race (S) of the same
species has chromosomes of equal length (Fig. IB). This chromosomal marker has proved to be
highly stable being present in all asexual cells (De Vries & Baguna, in preparation).
Nuclear size marker
Planarian tissues can be dissociated by maceration into single cells, the different cell types
characterized by phase-contrast microscopy, and their nuclear area measured (Baguna &
Romero, 1981). Neoblasts from biotypes A (diploid) and D (tetraploid) of Dugesia(S)polychroa
have mean nuclear areas of 25-76 ± 4-72pm2 (n = 100) and 41-17 ± 6-70/an2 (n = 100)
respectively, being thus easily distinguishable by size (Fig. 2A,B). This has proved to be a useful
marker, making possible to follow cell movement without studying karyotypes.
Cytoplasmic marker (fluorescent latex beads)
Fluorescent latex beads (Fluoresbrite carboxylate, Polysciences; green fluorescence, 1-Ojum in
diameter) were used to label the cytoplasm of some differentiated cell types (mainly
gastrodermal and fixed parenchyma cells). To do that, planarians were fed an artificial food
mixture made of beef liver, low-melting-point agarose (Sea Prep), andfluorescentlatex beads
(Romero, unpublished data). Once fed, the beads appear in gastrodermal cells, and 3-6 h later
they appear within some parenchymal cell types (fixed parenchyma, cianophilic cells,
acidophilic cells; Baguna & Romero, 1981). The beads remain there for at least 15-20 days,
disappearing slowly later on. No beads were seen to be taken up by undifferentiated cells
(neoblasts) probably due to their scanty cytoplasm. Labelled cells were detected by
epifluorescence (Leitz Dialux-20 microscope with a Ploemopak 2.4. equipment) (Fig. 3).
60
E. SAL6 AND J. BAGUNA
Grafting techniques
Grafting between As and S tissues of the same body level of Dugesia(S)mediterranea, between
diploid and tetraploid biotypes of Dugesia(S)polychroa, and between fluorescent-labelled graft
and unlabelled hosts of either species, were performed according to the classical procedures of
Dubois (1949) and Schilt (1974). To avoid bacterial contamination, all the experiments were
done under careful sterile conditions, and all the solutions employed had kanamycin sulphate
(Sigma) at lO/Ugml"1.
Irradiation
Organisms used to provide host irradiate tissues were exposed to 8000 rads (1000 radsmin"1)
using a HT-100 Philips X-ray machine (1-7mm Al Filter; 100kV, 8mA).
Measure of cell movement
Depending on the marker three methods were used:
1) Chromosomal marker (Fig. 4). Cell migration between As and S tissues of Dugesia
(S)mediterranea was measured by placing ten grafted organisms in 0-05 % colchicine (Sigma)
for 6 h at different periods after grafting (10,21,31 and 47 days). After, they were fixed in 1 NHC1, stained 'in toto' with a modified Gomori's method (Sal6 & Baguna, 1984), mounted
according to Baguna (1974), and all metaphase figures counted (according to Karyotypes of
As and S cells) along the anteroposterior (cephalocaudal) and mediolateral axis. From them,
the mitotic index of each kind of cell in 1 mm intervals along both axis was obtained. The rate
2A
v)
B
Fig. 2. (A) Neoblast of biotype A (diploid) of Dugesia(S)polychroa; (B) neoblast of
biotype D (tetraploid) of Dugesia(S)polychroa. Bar equals 5jum.
Fig. 3. Planarian fixed parenchyma cell with fluorescent latex beads (arrows) inside
the cytoplasm. Bar equals
Cell movement in planarians
61
of movement (in average jum/day) was calculated from the distance covered by the leading
edge of graft cells at different time intervals.
2) Nuclear marker (Fig. 5). To measure cell migration between diploid and tetraplpid biotypes
of Dugesia(S)polychroa, ten grafted organisms werefixedat different times after grafting (10,
21, 31 and 47 days), cut in 0-5 mm pieces along the anteroposterior axis, and macerated
according to the procedure of Baguna & Romero (1981). Neoblasts of both biotypes are
easily distinguishable in phase-contrast microscopy according to nuclear size. In each 0-5 mm
time
0-45 days
0
17
ant
U
post
V
bl
cut
time
0-45 days
V
Host
colchicine
+ fixation
+ staining
/T\
colchicine
+ fixation
+ staining
ant
- post
V
Fig. 4. Method used to measure the movement of graft cells within intact (upper right)
and regenerating (lower right) host of Dugesia(S)mediterranea using the chromosomal
marker, bl, blastema; g, graft; ant, anterior (cephalic); post, posterior (caudal). For
details, see text.
time
0-45 days
Donor
cut
time
0-45 days
Host
Fig. 5. Method used to measure the movement of graft cells within intact (upper right)
and regenerating (lower right) hosts of Dugesia(S)polychroa using the nuclear marker,
bl, blastema. For details, see text.
62
E. S A L 6 AND J. BAGUNA
interval, 10% of neoblasts (~1000 cells) were counted and classified according to their
biotype (A or D). The rate of graft cell movement (in average /um/day) was calculated from
the distance covered by the graft cells between different time intervals.
3) Cytoplasmic marker. Movement of fluorescent (green) labelled cells was measured by fixing
ten organisms at different times after grafting (5, 10, 15 and 20 days), mounting them 'in
toto', and observing by epifluorescence the edge of fluorescent cells. The rate of cell
movement (in average /an/day) was calculated from the distance covered by the leading edge
of fluorescent cells at different time intervals.
All measurements were made for unirradiated and irradiated intact and regenerating hosts.
RESULTS
Histology
a) Unirradiated hosts
Grafts of As or S tissue into S or As hosts of Dugesia(S)mediterranea take very
quickly. Two days after grafting, epidermal and mesodermal continuity is
complete and no scar or connective tissue formation at the graft-host boundary is
seen. Similar results were obtained with biotypes A and D of Dugesia(S)polychroa,
and with either species using the fluorescent marker.
b) Irradiated hosts
Grafts of unirradiated tissue into irradiated hosts in both species also take very
easily, epidermal and mesodermal continuity appearing quickly after grafting. As
expected, grafts of unirradiated tissue lead to survival and regeneration of the
irradiated host.
Cell movement in unirradiated intact organisms
The spreading of grafted As cells within S host tissues, and vice versa, in
unirradiated Dugesia(S)mediterranea is shown in Fig. 6A. Estimates of the average
rate of movement give values of 39-7 ± 11-0//m/day (n = 40) in both directions
(graft to host and host to graft). Since the only mitotic cells in planarians are
neoblasts, and assuming a mean cell diameter of 8-10//m for these cells, this rate
of movement is equivalent to 4 cell diameters. No differences are detected in the
direction of movement (spreading) along the A - P axis and mediolateral axis.
The rate of movement obtained using the difference in nuclear area as marker
between biotypes A and D of Dugesia(S)polychroa amounts to 42-2 ± 10-5 /im/day
(~4 cell diameters) (n=<40). On the other hand, fluorescent cells (all of them
differentiated) move (or spread) at a rate far slower than the one for undifferentiated cells: ^15/mi/day (n = 36). Since the mean cell diameter of
differentiated cells is ~30Jum (Baguna & Romero, 1981), this is equivalent to less
than half a cell diameter.
Cell movement in unirradiated regenerating organisms
Mutual cell spreading between graft and host tissues in regenerating
Dugesia(S)mediterranea is shown in Fig. 6B. The rate of cell movement is very close
63
Cell movement in planarians
10 days
0-3
31 days
21 days
47 days
rfrfl
i
•3 0-2
ant
g
post
ant
10 days
0-3
post
ant
post
ant
31 days
21 days
post
47 days
AM.
I 0-2
o
I 0-1
ant
g
post
ant
g
post
ant
g
post
ant
post
Fig. 6. Mitotic index ±S.D. of graft cells (hatched bars) and host cells (plain bars) by
interval of length (1 mm) along the anteroposterior (cephalocaudal) axis of intact (A),
and anteriorly regenerating (B) unirradiated hosts of Dugesia(S)mediterranea at
different times (in days) after grafting and cutting. Movement (spreading) of cells
occurs evenly in both directions, g; graft; ant, anterior (cephalic); post, posterior
(caudal).
to the one found for intact organisms (36-8 ± 6-9/im/day) (n = 42). Similar
experiments done with Dugesia(S)polychroa gave values (38-2±8-2jUm/day)
(n = 46) very close to those found for intact organisms. Moreover, the amount of
spreading is similar in all directions along the A - P axis and the mediolateral axis.
Fluorescent cells also move at a similar rate as in intact organisms (^15/im/day)
(0-5 cell diameter) (n = 40).
Cell movement in irradiated intact organisms
After irradiation at 8000 rads, planarian cells do not proliferate, cell renewal is
halted, tissues necrose, and organisms die within a 3-5 weeks period depending on
temperature, age, and nutritional conditions (Lange, 1968; Chandebois, 1976).
Moreover, the number of neoblasts decreases steadily approaching zero values at
15-20 days postirradiation (Salo, 1984).
Grafting unirradiated tissues into irradiated hosts leads to survival of the host
through repopulation from graft cells. That graft cells and not revitalized host cells
are responsible for this behaviour appear evident because all mitotic figures belong
to graft cells. The rate of movement of As-cells into irradiated S-hosts and of Scells into irradiated intact As-hosts in Dugesia(S)mediterranea gave values of
75-3 ± 12-4/an/day ( - 7 - 8 cell diameters) (n = 38) (Fig. 7A). This value is
64
E. SAL6 AND J. BAGUNA
significantly higher than the one found in similar experiments using irradiated intact hosts (Fig. 6A). Moreover, the spreading occurs evenly in all directions along
the A - P and mediolateral axis. Similar data were found using the nuclear size
marker of biotypes A and D of Dugesia(S)polychroa (70-23 ± 10-80 ^m/day) (7-8
cell diameters) (n = 40). As a control experiment, the spreading of As-cells grafted
10 days
0-3
31 days
21 days
45 days
0-2
A
Jo,
0
ant
\
g
post
ant
post
ant
post
ant
post
B
21 days
10 days
0-3
31 days
45 days
•a 0-2
o
rfi
O
1 0-1
0
. , r*
ant
post
ant
\
post
ant
g
post
ant
post
Fig. 7. Mitotic index ± S . D . of graft cells by interval of length (lmm) along the
anteroposterior (cephalocaudal) axis of intact (A) and anteriorly regenerating (B)
irradiated hosts of Dugesia(S)mediterranea at different times (in days) after grafting and
cutting. Movement (spreading) of cells occurs evenly in both directions, g, graft; ant,
anterior (cephalic); post, posterior (caudal).
10 days
21 days
45 days
0-3
•gO-2
01
ant
post
ant
post
ant
Fig. 8. Mitotic index ± S.D. and movement of graft cells along the anteroposterior
(cephalocaudal) axis of intact unirradiated (thick line and hatched bars) and irradiated
(thin line and plain bars) hosts at different times (in days) after grafting, g, graft; ant,
anterior (cephalic); post, posterior (caudal).
post
Cell movement in planarians
65
Table 1. Rates of cell movement (in pan/day) of graft cells within host tissues
(irradiated and non-irradiated; intact and regenerating) using chromosomal, nuclear,
and cytoplasmic markers
HOST
Irradiated
Unirradiated
Marker
Chromosomal
Nuclear
Cytoplasmic
Intact
Regenerating
Intact
Regenerating
39•7 ±11-0
42•2 ±10-5
36-8 ±6-9
38-2 ±8-2
75•3 ± 12-4
70•2 ± 10-8
sS 15
73-9 ±8-9
77-1 ± 10-4
into irradiated As-hosts was measured. The results obtained also gave similar
values (72-3 ± 11-85/im/day) (~7-8 cell diameters) (n = 46). However, using
fluorescent-labelled grafts, the spreading rate found (^15jUm/day; —0-5 cell
diameter) (n = 40) is similar to the one found for unirradiated organisms,
regenerating or not. This means that, unlike undifferentiated cells, differentiated
cells do not change their rate of spreading after irradiation.
Cell movement in irradiated regenerating organisms
The spreading of graft cells within irradiated regenerating hosts gave values very
close to the ones found for irradiated intact hosts: 73-9 ± 8-9//m/day (~7-8 cell
diameters) (n = 38) for Dugesia(S)mediterranea (Fig. 7B), and 77-06 ± 10-40/xm/
day (~7-8 cell diameters) (n = 42) for Dugesia(S)polychroa. The amount of
spreading was seen to be very similar in all directions along the A-P and
mediolateral axis. In addition, fluorescent cells migrate at a similar rate to that in
irradiated intact hosts (s$15jum/day; ~0-5 cell diameter) (n = 32).
To study why undifferentiated grafted cells move faster in irradiated than in
unirradiated hosts, the mitotic indices of grafted cells in both kinds of hosts were
compared. The results (Fig. 8, for Dugesia(S)mediterranea) show clearly a higher
mitotic index for graft cells within irradiated hosts than within unirradiated hosts.
Moreover, in irradiated hosts, the number of graft cells in mitosis at sites distant
from the graft site is higher than at the graft site itself, whereas in non-irradiated
hosts it is the opposite.
Table 1 summarizes for comparison the rates of cell movement found for all
experiments done and groups tested, using chromosomal, nuclear and cytoplasmic
markers.
DISCUSSION
The use of chromosomal, nuclear, and cytoplasmic markers to track cell movements in
planarians
Previous attempts to track cell movements within planarian tissues made use of
radioactively labelled cells and differences in pigmentation between graft and host
66
E. S A L 6 AND J. BAGUNA
strains. However, since planarian cells do not take up thymidine (Coward, Hirsh
& Taylor, 1970), the most commonly used labels were [3H]cytidine (Cecere,
Grasso, Urbani & Vannini, 1964), [3H]uridine (Lender & Gabriel, 1965), and
14
CO 2 (Flickinger, 1964), all of them labelling unspecifically RNAs and proteins of
most cell types. Once labelled, movement of cells was followed by autoradiography. The results found were diverse, a fact not surprising if we take into
account the differences in markers and techniques employed (see Br0nsted, 1969,
for general references). One of the main flaws of these experiments is that labelled
RNAs and proteins can, through cell lysis and diffusion into the intercellular
space, be made available to non-labelled cells and blur the results. Moreover,
these markers are non-specific, and non-permanent, being diluted with time up to
the point of no detection. On the other hand, the use of pigmentation differences
between planarian strains (or species) also has several disadvantages (see
Introduction).
The chromosomal and ploidy markers used in this work have several advantages over RNA/protein labelling and pigmentation markers: 1) they are
permanent; 2) they are present in all the cells of either graft or host; and 3) are of
easy identification. Moreover, the fluorescent marker, though non-permanent, has
proved very useful to follow specifically the movement of cells other than
neoblasts.
Cell movement within intact planarians
The ease with which graft and host cells mix suggest at first that cell movement
within planarian tissues is rather unrestrained, a fact not surprising if we consider
that parenchymal tissue in planarians is in a rather loose state of organization
(Baguna & Ballester, 1978). But, what kind of cell moves?
That neoblasts move is shown clearly in data on cell spreading based on mitotic
and non-mitotic cells (Table 1). Moreover, using fluorescent beads, it has been
shown that cells other than neoblasts (e.g. fixed parenchymal cells, acidophilic and
basophilic secretory cells, gastrodermal cells,..) move too, though their
movements are much more restrained. Why this is so is at present difficult to
assess, though it could be linked to their lack of cell proliferation. From this, we
can conclude, tentatively, that cell movement between graft and host is mainly due
to neoblasts.
Active cell movement or passive cell repopulation?
One of the uncertainties of the phenomenon of neoblast movement between
graft and host tissues is to know if this is due either to an active process of
individual cell movement (true cell migration) or to a passive process of 'cell
spreading' due to cell proliferation. The first alternative was suggested by Dubois
(1949) on the grounds that mitosis was not found within the irradiated host until
unirradiated cells or cells from the graft reached the wound.
The results found by us clearly contradict Dubois' proposal. First of all, many
graft cells in mitosis are found within unirradiated and irradiated hosts soon after
Cell movement in planarians
61
grafting. Secondly, the mean rates of movement found (35-75 /xm/day) are rather
low for a cell often qualified as a 'migratory cell', values that can be easily
accounted by random movements of the daughter cells during and after telophase.
Finally, similar rates of movement are found using the chromosomal marker (seen
in mitotic neoblasts) and the nuclear size marker (seen in non-mitotic neoblasts),
indicating that non-mitotic neoblasts do not move at a faster rate than mitotic
neoblasts. The last two points clearly suggest that the spreading of neoblasts
should be mainly linked to cell proliferation, and more specifically to random
movements occurring during and/or after cell division.
This leads us to conclude that, as already suggested by Sugino, Okuno &
Yoshinobu (1970), movement of cells (neoblasts) from graft to host and vice versa
is not the result of active cell movements of individual cells but the consequence of
a passive process of cell spreading (repopulation) mainly linked to cell division.
Cell movement from distant sites is neither stimulated by the wound nor directed
towards it
That wounding does not stimulate the spreading of cells far from the wound
appears very clear when comparing the similar rate of movement of neoblasts in
regenerating and intact organisms (Table 1). Moreover, that cells other than
mitotic cells do not spread faster after wounding is also clear when looking at the
data obtained using the nuclear and the cytoplasmic markers (Table 1).
These data contradict one of the tenets of Wolff and Dubois' theory of blastema
formation: that wounding stimulates the 'migration' of cells even in regions far
from the wound, stimulation mediated by some kind of diffusable 'necrohormone'
released at or near the wound. Besides the results found in this work, we think
their conclusions are unproved for two reasons: 1) in their experiments they did
not have rates of movement in intact organisms to compare with the values found
for regenerating organisms and infer from the latter only that stimulation had
occurred; and 2) the existence of the postulated 'necrohormones', or any other
stimulatory mechanism, have never been substantiated.
If spreading of cells is not stimulated in regenerating organisms, it follows that
anteriorly regenerating organisms should have similar spreading rates of cells
towards the wound (anterior) than towards posterior intact areas. That this is so
appears very clear from all the data gathered using both markers (chromosomal
and nuclear), where an even rate of spreading was seen along the A-P axis (Figs
6A,B, 7A,B) and the mediolateral axis (results not shown) in all groups studied.
From this we can conclude that, contrary to another of the tenets of Wolff
and Dubois' theory, cell movement (spreading) is not directed (oriented)
preferentially to the wound.
Why cells move faster in irradiated than in unirradiated hosts?
The rate of movement of grafted cells in irradiated hosts is twice the value found
for unirradiated hosts (Table 1). This could be due either to an easier movement of
graft cells going through the spaces left by lysed or dying cells, or to a higher
68
E. S A L 6 AND J. BAGUNA
mitotic rate of graft cells in irradiated hosts due to the decreasing number of
neoblasts and to the absence of mitosis in the latter.
The mitotic index of graft cells in irradiated hosts is two to four times higher
than in unirradiated hosts (Fig. 8). Moreover, the rate of cell movement measured
using the chromosomal marker (seen in mitotic neoblasts) and the nuclear size
marker (seen in non-mitotic neoblasts) are similar (Table 1). Both results favour
the second alternative; that is, that absence of mitotic cells in the irradiated host
and the ever decreasing number of host neoblasts allow graft cells to engage in
mitosis at a rate higher than in unirradiated host. From this it follows that cell
proliferation in planarians could be controlled by a density-dependent inhibitory
mechanism mediated by factors produced and released by neoblasts themselves.
This, jointly with the action of some still poorly characterized factors that trigger
neoblast proliferation after wounding (Baguna, 1976; Friedel & Webb, 1979; Sal6,
1984) and feeding (Baguna, 1974), may control the final balance of neoblast
density and proliferation.
Some implications for mechanisms of blastema formation and the origin of blastema
cells
In a previous paper (Salo & Baguna, 1984) it has been shown that regenerative
blastemata in planarians do not have mitotic activity, and that their growth could
be explained through the continuous entrance of undifferentiated cells from the
stump to the base of blastemata. However, it is still uncertain if these cells come
only from local stump sources or if cells placed far from the wound can participate
m blastemata growth.
The low spreading rates of planarian cells found in this work (see Table 1)
suggest at first a local origin of blastema cells. Thus, taking into account that
mitotic activity in the stump region near the wound is three to four times higher
than in control intact organisms (Salo & Baguna, 1984), and assuming that cell
spreading is mainly due to movements related to mitotic activity, it is sound to
suggest that spreading rates in regions near the wound may attain values around
100 /im/day (recent unpublished data obtained on cell spreading near the wound
support this suggestion; Salo & Baguna, in preparation). If this is so, a 4- to 5-day
blastema must be the result of the proliferation and spreading of cells within a
region of, at the most, 300-500 /im around the wound. This means that, in normal
(non-irradiated) regenerating organisms, blastema cells have a local origin.
However, as first shown by Wolff & Dubois (1948), partially irradiated
regenerating organisms form a blastema after a delay proportional to the distance
between wound and healthy unirradiated tissue. From this they suggested that
blastema cells were neoblasts that had migrated throughout the irradiated tissue
from the unirradiated regions, and, therefore, that neoblasts were totipotent or
pluripotent migratory cells.
The results obtained in this work suggest an alternative interpretation of Wolff
and Dubois experiments and conclusions. First of all, the so-called 'migration' of
neoblasts is not a true cell migration but the result of the slow and progressive
Cell movement in planarians
69
spreading of undifferentiated cells (neoblasts) through the irradiated region by cell
proliferation. Secondly, although neoblasts from the unirradiated region make the
blastema in partially irradiated organisms, this does not necessarily mean that
neoblasts are totipotent or pluripotent since these cells (neoblasts) may have
resulted from the dedifferentiation of differentiated cells in the unirradiated
region before their 'migration' (spreading) to the wound. Therefore, until more
precise experiments using cell-specific markers are performed (Salo & Baguna,
work in progress), the idea of neoblasts as totipotent or pluripotent cells will
remain unproved.
We would like to thank Rafael Romero for permission in quoting unpublished data, Professor
N. G. Lepori (Universita di Sassari, Sardegna, Italia) for sending stocks of the sexual race of
Dugesia(S)mediterranea and the biotypes A and D of Dugesia(S)polychroa, and Dr J. Schilt
(University de Nancy I, France) for teaching one of us (ES) grafting techniques in planarians.
We warmly acknowledge the comments of an anonymous referee which greatly helped to
improve the manuscript. This work was supported by the grants AIUB 611/81 of the University
of Barcelona and 1108/81 from Comisi6n Asesora de Investigaci6n Cientifica y Tecnica
(CAICYT) to JB.
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(Accepted 5 March 1985)